Antibodies to Complement Pathway Components

Preclinical studies using animal models and human derived samples demonstrate that inhibition of complement ameliorates many inflammatory and autoimmune disease manifestations. The AP pathway has been shown to play a particular important role (for review of AP pathway in various diseases see Holers “The spectrum of complement altnerative pathway-mediated diseases, Immunological Revies, 2008).  

Factor B: 

Factor B is a 90 kDa single chain glycoprotein composed of 5 protein domains. The amino terminal region (Ba) consists predominantly of 3 short consensus repeats (SCRs) domains found in complement regulatory proteins. The carboxy-terminal region (Bb) consists of a type A domain found in von Willebrand factor and complement receptors, followed by a trypsin like serine protease domain. Hourcade (J. Biological Chemistry, 270(34), 1995, pp. 19716-19722. 

Monoclonal antibodies to human factor B which prevent factor B binding to C3b are known (Clardy, “Complement Activation by whole endotoxin is blocked by a monoclonal antibody to Factor B. Infect. and Immun. 1994, 62, 4549-4555).

Bansal (US7,959,919B2, US2005/0107319 and US7,959,919) discloses a method of inhibiting complement activation in the treatment of vsirous disorders by using factor B inhibitors such as antibodies to human factor B. 

–Factor Ba: 

Hourcade (J. Biological Chemistry, 270(34), 1995, pp. 19716-19722) disclose using site directed mutagenesis of recombinant factor B and monoclonal antibody epitope mapping to identify 3 sites on factor Ba; site 1 which is a stretch of 19 contiguos amino acids in short consensus repeat 1 that forms the epitope of a monoclonal antibody that effectively blocks factor B function, site 2, composed of a contiguous amino acids in short connsensus repeat 2 and site 3, consisting of 7 contiguous amino acids in short consensus repeat 3 were also defined by mutations that reduced factor B hemolytic activity ot 3% or less. Further analyses indicated that sites 2 and 3 contribute to factor B-C3b interactions. 

Bansal (US12501165) discloses a method of inhibiting factor Ba dependent complement activation in bood of a subject by administering anti-factor Ba antibodies which specifically bind to factor Ba protein sequences involved in Factor B binding to C3b. 

The protein Ba (cleaved from Factor B) is a large protein. Any of a multiude of antiboides can be produced against various protein motifs of, and locations on, the protein (Bansal, US13/646286). 

Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995) discloses an anti-Ba mAb that blocks factor B hemolytic activity and appears to interfere with the normal binding of factor B to C3b. 

Tanaka (Immunology, 73, 1991, pp.383-387) discloses a murine monoclonal antibody directed against the Ba domain of factor B that enhanced binding of intact factor B with cell bound C3b and the haemolytic activity of factor B. 

Ueda (J. Immunology) 138(4), 1143-1149, 1987) discloses two antibodies that react with distinct epitopes on Ba and inhibited the hemolytic activity of B. The two anti-Ba Mabs also inhibited binding of B to C3b, suggesting that the binding site for C3b on intact B is located on the Ba portion of the molecule. 

–Factor Bb: 

Bansal (US12/675220, published as US2010/0239573) teaches a method of inhibiting AP by administering antibodies that specifically binds to an epitope of the Bb segment of factor B. 

Ueda (J. Immunology) 138(4), 1143-1149, 1987) discloses two antibodies which reacted with the same or adjacent peptides on the Bb fragment of B and inhibited the hemolytic activity of B. 

Factor D antibodies: The following patents disclose inhibition of complement activation using factor D antibodies; Fung (US 81,124,090, US8,236,317B2; see also US2002/0081293). Ann (US 8,193,329B2).

The following applications disclose anti-factor D antibodies for the treatment of complement associated eye conditions such as chroidal neovascularization (CNV) and age-related macular degeration (AMD) (Fung US application # 12/092346; Hass US 8268310)

Tanhehco (Transplantation Proceedings, 31, 2168-2171 (1999) discloses an anti factor D antibody, MAb 166-32 which inhibits the AP.

For anti-factor D antibodies and cardiopulmonary Bypass see Undar (Ann Thorac Surf 2002, 74, 355-62). 

C3b: is a large protein and thus multiple antibodies can be produced against various segments of this protein. Tthere exists multiple sites where an antibody might bind and inhibit the protein’s activity in any variety of ways. Depending on how and where an antibody binds to C3b, the effect of that antibody could range from inconsequential to complete inhibition (Bansal, US13/646286). 

Bansal (US2010/0111946) discloses the use of anti-C3b antibody to inhibit C3 dependent complement activation. The anti-C3b antibody specifically binds to C3b protein sequences involved in C3b binding to properdin or Factor B or C3b cleavage from C3. 

DiLillo (Mol Immunol., 2006, 43(7): 1010-9) discloses selective inhibition of the AP by an anti-C3b/iC3b monoclonal antibody and by a chjimeric, deimmunized form of the antibody. 

Taylor (WO2006/012621) discloses the use of an antibody directed against C3b as a means of inhibiting the AP. 

C5: 

Eculizumab and pexelizumab® (Soliris®; Alexion Pharmaceuticals) are FDA approved anti-C5 antibodies.  

Antibodies which bind to complement C5 and prevent C5 form being cleaved are disclosed in US Patent No: 6,355,245 and include both a whole or full lenght antibody (eculizumab) and a single chain antibody (pexelizumab). A similar antibody against mouse C5 is called BB5.1 (see Rother, WO2005/110481).

Eculizumab is an approved antibody directed against complment compoonent C5 for the disease paroxysmal nocturnal hemoglobinuria.

Rother (WO2010/054403) discloses inhibitors of C5 such as a C5 antibody and methods for using the compositions to treat complement assocaited disorders. 

Evans (US6,355,245) beaches using anti-C5 antibodies to treat glomerulonephritis.

Zhou (US2014/0056888) teaches highly concentration aqueous solutions of an anti-C5 antibody which can be advantageously administered to a patient in a small volume. 

Methods for identifying the epitope to which a particular antibody binds are known in the art. For example, the binding epitope of an anti-C5 antibody can be identified by measuring the binding of the antibody to several overlapping epptide fragments of a C5 protein. Each of the different overlapping pepties is then bound to a unique address on a solid support (e.g., separate wells of a multi-well assay plate). Next, the anti-C5 antibody is interrogated by contacting it to each of the peptides. Unbound anti-C5 antibody is removed by washing. Next, a detectably-labeled secondary antibody that binds to the anti-C5 antibody, if present in a well of the plate is contacted to each of the wells and unbound secondary antibody is removed by washing. The presenceof the detectable signal produced by the detetably-labeled secondary antibody in a well is an indicaiton that hte anti-C5 antibody binds to the particular peptide fragment assocaited with the well. A particular epitope to whcih the antibody binds can also be identified usign BIAcore chromatographic techniques. (Rother, (PCT/US2009/063929)

–C5a: Synthetic peptides derived from C5a are known antagonists of C5a. However, the antagonistic activity is highly dependent upon the structure of the peptides. For example, whereas a 14 amino acid residue peptide from the C5a C-terminal region can function as an antagonist of C5a, 5-8 amino acid residue peptide analogues of the same region have been reported to be agonists rather than antagonists (Kaneko, Immunology, 1995, 86, 149-154).

Wang (US7,279,158) dicloses use of compounds such as antibodies that block complement component C5 to treat established joint inflammation (arthritis).

–C5a receptor (CD88): Several antagonists for C5a receptor (CD88) have been described, including peptides, a non-peptidic compound, C5a mutants and anti-C5R-antibodies. All of these compounds are potent CD88 antagonists in vitro; however, only the C5a mutants C5aRAM and jun/fos-A8, the cyclic peptide AcPhe[AcF-(pdChaWR), and a nonpeptidc antagonist have been proven useful in vivo (Otto, J. Biological Chemistry, 279, 1, 2004, 142-151).

–C5b antibodies: are known in the art (Evans, US6,355,245) and commercially available anti-C5b antiboides are available. 

Properdin antibodies: (see also Properdin under “complement” for structure of properdin)

Brunden and Bansal disclose anti-human properdin MoAbs which blocked activation of the AP in vitor and in ex vivo models of CPB (Molecualr Immunology, 36, No. 4-5). 

Gupta-Bansal discloses an anti-properdin antibody which caused nearly complete inhibition of the C3a and C5b-9 formation in a cardiopulmonary bypass model (Molecular Immunology 37 (2000) 191-201). 

Gupta-Bansal discloses a number of patents claiming anti-properdin antibodies: US6,333,034, issued 12/25/2001 (inhibit AP); US 2003/0198636 (US 6,333,034, issued 12/25/2001) (US2002/0015701) (US7,423,128, issued 9/9/2008) (US2006/0093599 (binds epitope within a thrombospondin type 1 repeat of propderin)); US13/849092 (anti-properdin antibody useful for inhibiting AP that is specific to amino acid sequence at N-terminal end of properdin). 

Holers (US2006/0292141) teaches antagonists to complement proteins such as properdin. Holers teaches that properdin has the sequence

Song (US12/663,690, US 2010/0263061; see also PCT/US08/07270 and WO/2008/154018) also disclose anti-properdin mAbs which selectively inhibit AP and have no effect on the AP amplifcation loop of the CP.

Bansal discloses (US 12/920,997, US 2011/008340, now US 8,435, 512; see also PCT/US2008/068530; see also US 13/849092 (also teaching anti properdin antibody that competitively inhibits binding of an antibody to a proeprdin sequence)) discloses an antibody that specifically binds to an epitope of the N terminus end of properdin that blocks the alternative pathway activaiton without affecitng the classical pathway activation. See also US8,664,362 and 14/195458 disclosing anti-properdin antibody that can bind to the N-terminal domain of properdin to inhibit the bidning of properdin to C3b. 

Properdin is a large protein with many potential sites where antibodies can bind. Not all, or even most, of these antibodies will necessarily have any therapeutic value (Bansal, US13/646286). 

Antibodies to Complement Regulatory Proteins

CD59: CD59 inhibits MAC formation by specifically binding to complement proteins C8alpha and C9. Giddings (Nature Structural & Molecular Biology, 11(12), 2004). Qin (US 13/391124) discloses antibodies that bind to CD59, inhibiting binding of CD59 to C8 and/or C9. The antibodies bind the same epitope of CD59 as domain 4 of intermedilysin (ILYd4). The antibodies were identified by taking erythrocytes from humans or hCD59RBC trasngenic mice that express only human CD59 and incubating them with a candidate antibody, then incubation with ILYd4 or mouse anti-hCD59, followed by FITC conjugate anti-His antibody or FITC-conjugated secondary antibody. Cells incubated with antibodies that bound to the same epitope as ILY4 did not stain with either ILYd4 plus FITC anti-HIS or FITC secondary antibody. 

Qin (US13391124) disclose antibodies that bind to CD59 and inhibit binding of CD59 to complement proteins C8 and C9. The antibodies bind to the same epitope as ILYYd4 and thus inhibit binding of ILYd4 to CD59. Intermedilysin (ILY) is a cytoxin secreted by Streptococcus intermedius which lyses only human cells due to its receptor specificity for human CD59 via its domain 4 (ILYd4). ILYd4 binds to AA42-58 in hCD59, which also participates in the binding to C8 and C9. A truncated ILYd4 has been shown to abrogate hCD59 function to facilitate CDC effect on cancer and HIV. Hu (“Domain 4 of ILY sensitizes antibody therapy on cancer and HIV through abrogating human CD59 function”) showed ILYd4 sensitized a Rituximab-resistance B lymphoma cells to Rituximab treatment without toxicity effects and sensitized HIV to complement mediated virolysis activated to HIV patient’s antibody. The alpha helical region, specifically, residues 42-58 of huCD59 contains the ILY-binding site. This same region is also recognized and bound by complement proteins C8alpha and C9. Thus the domain 4 of ILY and complement proteins C8alpha and C9 recognize the same or overlapping regions of huCD59. Giddings (Nature Structural & Molecular Biology, 11(12), 2004) disclsoses an antibody (10G10) that completely blocked ILY binding and cytolytic activity to huCD59. 

Sims (US2003/0166565) teaches compounds such as an antibody which specifically bind to amino acids 42-58 of huCD59 and thereby block CD59 binding to C9 (¶s17, 41; claims 28-29).